Novel anti-igf-ir antibodies and uses thereof

ABSTRACT

The present invention relates to novel antibodies capable of binding specifically to the human insulin-like growth factor I receptor IGF-IR and/or capable of specifically inhibiting the tyrosine kinase activity of said IGF-IR, especially monoclonal antibodies of marine, chimeric and humanized origin, as well as the amino acid and nucleic acid sequences coding for these antibodies. The invention likewise comprises the use of these antibodies as a medicament for the prophylactic and/or therapeutic treatment of cancers overexpressing IGF-IR or any pathology connected with the overexpression of said receptor as well as in processes or kits for diagnosis of illnesses connected with the overexpression of the IGF-IR. The invention finally comprises products and/or compositions comprising such antibodies in combination with anti-EGFR antibodies and/or anti-VEGF antibodies and/or antibodies directed against other growth factors involved in tumor progression or metastasis and/or compounds and/or anti-cancer agents or agents conjugated with toxins and their use for the prevention and/or the treatment of certain cancers.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of copending application Ser. No.12/973,480 filed on Dec. 20, 2010, which is a Divisional of applicationSer. No. 11/658,739 filed on Jan. 29, 2007, now U.S. Pat. No. 7,854,930,which issued Dec. 21, 2010, and which is a National Phase of PCTInternational Application No. PCT/IB2005/002619 filed on Jul. 27, 2005,which claims the benefit of U.S. Provisional Application No. 60/591,932filed on Jul. 29, 2004, and to Patent Application No. 0408379 filed inFrance, on Jul. 29, 2004. The entire contents of all of the aboveapplications are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

The present invention relates to novel antibodies capable of bindingspecifically to the human insulin-like growth factor I receptor IGF-IRand/or capable of specifically inhibiting the tyrosine kinase activityof said IGF-IR, especially monoclonal antibodies of murine, chimeric andhumanized origin, as well as the amino acid and nucleic acid sequencescoding for these antibodies. The invention likewise comprises the use ofthese antibodies as a medicament for the prophylactic and/or therapeutictreatment of cancers overexpressing IGF-IR or any pathology connectedwith the overexpression of said receptor as well as in processes or kitsfor diagnosis of illnesses connected with the overexpression of theIGF-IR. The invention finally comprises products and/or compositionscomprising such antibodies in combination with anti-EGFR antibodiesand/or anti-VEGF antibodies and/or antibodies directed against othergrowth factors involved in tumor progression or metastasis and/orcompounds and/or anti-cancer agents or agents conjugated with toxins andtheir use for the prevention and/or the treatment of certain cancers.

The insulin-like growth factor I receptor called IGF-IR is a welldescribed receptor with tyrosine kinase activity having 70% homologywith the insulin receptor IR. IGF-IR is a glycoprotein of molecularweight approximately 350,000.

It is a hetero-tetrameric receptor of which each half-linked bydisulfide bridges is composed of an extracellular α-subunit and of atransmembrane β-subunit. IGF-IR binds IGF1 and IGF2 with a very highaffinity (Kd #1 nM) but is equally capable of binding to insulin with anaffinity 100 to 1000 times less. Conversely, the IR binds insulin with avery high affinity although the IGFs only bind to the insulin receptorwith a 100 times lower affinity. The tyrosine kinase domain of IGF-IRand of IR has a very high sequence homology although the zones of weakerhomology respectively concern the cysteine-rich region situated on theα-subunit and the C-terminal part of the β-subunit. The sequencedifferences observed in the α-subunit are situated in the binding zoneof the ligands and are therefore at the origin of the relativeaffinities of IGF-IR and of IR for the IGFs and insulin respectively.The differences in the C-terminal part of the β-subunit result in adivergence in the signalling pathways of the two receptors; IGF-IRmediating mitogenic, differentiation and anti-apoptosis effects, whilethe activation of the IR principally involves effects at the level ofthe metabolic pathways (Baserga et al., Biochim. Biophys. Acta,1332:F105-126, 1997; Baserga R., Exp. Cell. Res., 253:1-6, 1999).

The cytoplasmic tyrosine kinase proteins are activated by the binding ofthe ligand to the extracellular domain of the receptor. The activationof the kinases in its turn involves the stimulation of differentintra-cellular substrates, including IRS-1, IRS-2, Shc and Grb 10(Peruzzi F. et al., J. Cancer Res. Clin. Oncol., 125:166-173, 1999). Thetwo major substrates of IGF-IR are IRS and Shc which mediate, by theactivation of numerous effectors downstream, the majority of the growthand differentiation effects connected with the attachment of the IGFs tothis receptor. The availability of substrates can consequently dictatethe final biological effect connected with the activation of the IGF-IR.When IRS-1 predominates, the cells tend to proliferate and to transform.When Shc dominates, the cells tend to differentiate (Valentinis B. etal., J. Biol. Chem. 274:12423-12430, 1999). It seems that the routeprincipally involved for the effects of protection against apoptosis isthe phosphatidyl-inositol 3-kinases (PI 3-kinases) route (Prisco M. etal., Horm. Metab. Res., 31:80-89, 1999; Peruzzi F. et al., J. CancerRes. Clin. Oncol., 125:166-173, 1999).

The role of the IGF system in carcinogenesis has become the subject ofintensive research in the last ten years. This interest followed thediscovery of the fact that in addition to its mitogenic andantiapoptosis properties, IGF-IR seems to be required for theestablishment and the maintenance of a transformed phenotype. In fact,it has been well established that an overexpression or a constitutiveactivation of IGF-IR leads, in a great variety of cells, to a growth ofthe cells independent of the support in media devoid of fetal calfserum, and to the formation of tumors in nude mice. This in itself isnot a unique property since a great variety of products of overexpressedgenes can transform cells, including a good number of receptors ofgrowth factors. However, the crucial discovery which has clearlydemonstrated the major role played by IGF-IR in the transformation hasbeen the demonstration that the R− cells, in which the gene coding forIGF-IR has been inactivated, are totally refractory to transformation bydifferent agents which are usually capable of transforming the cells,such as the E5 protein of bovine papilloma virus, an overexpression ofEGFR or of PDGFR, the T antigen of SV 40, activated ras or thecombination of these two last factors (Sell C. et al., Proc. Natl. Acad.Sci., USA, 90:11217-11221, 1993; Sell C. et al., Mol. Cell. Biol.,14:3604-3612, 1994; Morrione A. J., Virol., 69:5300-5303, 1995; CoppolaD. et al., Mol. Cell. Biol., 14:4588-4595, 1994; DeAngelis T. et al., J.Cell. Physiol., 164:214-221, 1995).

IGF-IR is expressed in a great variety of tumors and of tumor lines andthe IGFs amplify the tumor growth via their attachment to IGF-IR. Otherarguments in favor of the role of IGF-IR in carcinogenesis come fromstudies using murine monoclonal antibodies directed against the receptoror using negative dominants of IGF-IR. In effect, murine monoclonalantibodies directed against IGF-IR inhibit the proliferation of numerouscell lines in culture and the growth of tumor cells in vivo (Arteaga C.et al., Cancer Res., 49:6237-6241, 1989; Li et al., Biochem. Biophys.Res. Com., 196:92-98, 1993; Zia F. et al., J. Cell. Biol., 24:269-275,1996; Scotlandi K. et al., Cancer Res., 58:4127-4131, 1998). It haslikewise been shown in the works of Jiang et al. (Oncogene,18:6071-6077, 1999) that a negative dominant of IGF-IR is capable ofinhibiting tumor proliferation.

Cancer pathologies are characterized by an uncontrolled cellular growth.In several cancer, growth factors are specifically binding with theirreceptors and then transmit growth, transformation and/or survivalsignals to the tumoral cell. The growth factor receptors over-expressionat the tumoral cell surface is largely described (Salomon D. S. et al.,Crit. Rev. Oncol. Hematol., 1995, 19:183; Burrow S. et al., J. Surg.Oncol., 1998, 69:21; Hakam A. et al., Hum. Pathol., 1999, 30:1128; RailoM. J. et al., Eur. J. Cancer, 1994, 30:307; Happerfield L. C. et al., J.Pathol., 1997, 183:412). This over-expression, or abnormal activation,leading to a direct perturbation of cellular growth regulationmechanisms, can also affect the cell sensibility to induced apoptose byclassical chemotherapies or radiotherapies.

During last few years, it has been shown that the targeting of growthfactor receptors, like EGFR or Her2/neu over-expressed on the tumoralcell surface, with respectively humanized (Herceptin®) or chimeric(C225) antibodies results in an significant inhibition of the tumoralgrowth in patients and in a significant increase of the efficacity ofclassical chemotherapy treatments (Carter P., Nature Rev. Cancer, 2001,1(2):118; Hortobagyi G. N., Semin. Oncol., 2001, 28:43; Herbst R. S. etal., Semin. Oncol., 2002, 29:27). Other receptors like IGF-IR or VEGF-R(for vascular endothelial growth factor receptor) have been identifiedas potential target in several preclinical studies.

More particularly, IGF-IR is part of the tyrosine kinase receptors. Itshows a high homology with the Insulin receptor (IR) which exist undertwo isoforms A and B.

Sequences of IR, isoforms A and B, are registered under AccessionNumbers X02160 and M10051, respectively, in the NCBI Genbank. Otherdata, without limitations, relating to IR are incorporated herein byreferences (Vinten et al., 1991, Proc. Natl. Acad. Sci. USA, 88:249-252;Belfiore et al., 2002, The Journal of Biological Chemistry,277:39684-39695; Dumesic et al., 2004, The Journal of Endocrinology &Metabolism, 89(7):3561-3566).

The IGF-IR and IR are tetrameric glycoproteins composed of twoextracellular α- and two transmembrane β-subunits linked by disulfidebonds. Each α-subunit, containing the ligand-binding site isapproximately 130- to 135-kDa, whereas each (3-subunit containing thetyrosine kinase domain is approximately 90- to 95-kDa. These receptorsshare more than 50% overall amino acid sequence similarity and 84%similarity in the tyrosine kinase domain. After ligand binding,phosphorylated receptors recruit and phosphorylate docking proteins,including the insulin receptor substrate-1 protein family (IRS1), Gab1and Shc (Avruch, 1998, Mol. Cell. Biochem., 182, 31-48; Roth et al.,1988, Cold Spring Harbor Symp. Quant. Biol. 53, 537-543; White, 1998,Mol. Cell. Biochem., 182, 3-11; Laviola et al., 1997, J. Clin. Invest.99, 830-837; Cheatham et al., 1995, Endocr. Rev. 16, 117-142), leadingto the activation of different intracellular mediators. Although boththe IR and IGF-IR similarly activate major signalling pathways,differences exist in the recruitment of certain docking proteins andintracellular mediators between both receptors (Sasaoka et al., 1996,Endocrinology 137, 4427-4434; Nakae et al., 2001, Endocr. Rev. 22,818-835; Dupont and Le Roith 2001, Horm. Res. 55, Suppl. 2, 22-26; Kovalet al., 1998, Biochem. J. 330, 923-932). These differences are the basisfor the predominant metabolic effects elicited by IR activation and thepredominant mitogenic, transforming and anti-apoptotic effects elicitedby IGF-IR activation (De Meyts et al., 1995, Ann. N.Y. Acad. Sci., 766,388-401; Singh et al., 2000, Prisco et al., 1999, Horm. Metab. Res. 31,80-89; Kido et al. 2001, J. Clin. Endocrinol. Metab., 86, 972-979).Insulin binds with high affinity to the IR (100-fold higher than to theIGF-IR), whereas insulin-like growth factors (IGF1 and IGF2) bind to theIGF-IR with 100-fold higher affinity than to the IR.

The human IR exists in two isoforms, IR-A and IR-B, generated byalternative splicing of the IR gene that either excludes or includes 12amino acid residues encoded by a small exon (exon 11) at thecarboxy-terminus of the IR α-subunit. The relative abundance of IRisoforms is regulated by tissue specific and unknown factors (Moller etal., 1989, Mol. Endocrinol., 3, 1263-1269; Mosthaf et al., 1990, EMBOJ., 9, 2409-2413). IR-B is the predominant IR isoform in normal adulttissues (adipose tissue, liver and muscle) that are major target tissuesfor the metabolic effects of insulin (Moller et al., 1989; Mosthaf etal., 1990). IR-A is the predominant isoform in fetal tissues andmediates fetal growth in response to IGF2 (Frasca et al., 1999, Mol.Cell. Biol., 19, 3278-3288), as also suggested by genetic studiescarried out in transgenic mice (DeChiara et al., 1990, Nature 345,78-80; Louvi et al., 1997, Dev. Biol. 189, 33-48). Moreover, when cellstransform and become malignant, dedifferentiation is often associatedwith an increased IR-A relative abundance (Pandini et al., 2002, TheJournal of Biological Chemistry, Vol. 277, N^(o) 42, pp 39684-39695).

Given the high degree of homology, the insulin and IGF-I half-receptors(composed of one α- and one β-subunit) can heterodimerize, leading tothe formation of insulin/IGF-I hybrid receptors (Hybrid-R) (Soos et al.,1990, Biochem J., 270, 383-390; Kasuya et al., 1993, Biochemistry 32,13531-13536; Seely et al., 1995, Endocrinology 136, 1635-1641; Bailyeset al., 1997, Biochem J. 327, 209-215).

Both IR isoforms are equally able to form hybrids with IGF-IR. Hybrid-R,however, have different functional characteristics. Hybrid-RsB hasreduced affinity for IGF1 and especially for IGF2. In contrast,Hybrid-RsA has a high affinity for IGF1 and bind also IGF2 and insulinat a physiological concentration range. The expression of Hybrid-RsAup-regulates the IGF system by two different mechanisms i) binding (withhigh affinity) and activation by both IGF1 and IGF2 (which do not occurwith the Hybrid-RsB), ii) activation of the IGF-IR pathway after insulinbinding. Insulin binding to Hybrid-RsA phosphorylates the IGF-IRβ-subunit and activates an IGF-IR-specific substrate (CrkII) so thatHybrid-RsA shifts insulin to IGF-IR signaling (Pandini et al., 2002).

In several tissues, like liver, spleen or placenta, Hybrid-R are morerepresented than IGF-IR (Bailyes et al., 1997). As tumor tissuesoverexpress, or present an abnormal activation, both IGF-IR and IR-A(Frasca et al., 1999; Sciacca et al., 1999, Oncogene 18, 2471-2479;Vella et al., 2001, Mol. Pathol., 54, 121-124), Hybrid-RsA may also beoverexpressed in a variety of human malignancies, including thyroid andbreast cancers providing a selective growth advantage to malignant cellsable to respond by a type IGF-IR signalisation following a stimulationby IGF1 and/or IGF2 but also by insulin at physiological concentrations(Bailyes et al., 1997; Pandini et al., 1999, Clin. Cancer Res., 5,1935-1944; Belfiore et al., 1999, Biochimie (Paris) 81, 403-407; Frascaet al. 1999, Sciacca et al., 1999; Vella et al., 2001).

The realisation of such “therapeutic tools” able to block in the sametime the two receptors is of particular interest as they will allow toavoid the escape phenomena mediated by the expression, or abnormalactivation, in a same tumor of IGF-IR and hybrid-R.

Regarding the increasing interest on IGF-IR and, more particularly,monoclonal antibodies able to bind to, or inhibit the tyrosine kinaseactivity of, IGF-IR, the applicants have already developed andcharacterized a humanized monoclonal antibody called 7C10 or h7C10(coded F50035). An international patent application PCT/FR 03/00178relating to this antibody and its uses have been filed and published on24 Jul. 2003 under the publication number WO 03/059951. The content ofthis patent application is incorporated herein by reference.

BRIEF SUMMARY OF THE INVENTION

The object of the present invention is to be able to have availableother murine monoclonal antibodies, preferably chimerized or humanizedantibodies, which will recognize IGF-IR specifically and with greataffinity. These antibodies will interact little or not at all with theIR. Their attachment will be able to inhibit in vitro the growth oftumors expressing IGF-IR by interacting principally with the signaltransduction pathways activated during IGF1/IGF-IR and IGF2/IGF-IRinteractions. These antibodies will be able to be active in vivo on allthe types of tumors expressing IGF-IR including estrogen-dependenttumors of the breast and tumors of the prostate.

The present invention also allows to jointly block the hybrid-R andIGF-IR activity by generating a compound, and more particularlyantibodies, of high affinity able to bind to said two receptors and alsoto block their activation by IGF1, IGF2 or Insulin.

The present invention also deals with the use of isolated antibodiesaccording to the present invention, or a fragment thereof, saidantibodies or fragment being able to bind to i) human IGF-IR, and/or toinhibit the binding of its native ligands, preferably IGF1 and/or IGF2,and/or also able to inhibit specifically the tyrosine kinase activity ofsaid IGF-IR and/or ii) hybrid-R, and/or to inhibit the binding of itsnative ligands, preferably IGF1, IGF2 and/or Insulin, and/or also ableto specifically inhibit the tyrosine kinase activity of said hybrid-R.

According to another preferred embodiment, said antibodies are used forcancer therapy, more particularly breast cancer therapy.

Actually, it is known that breast tumoral cells specifically present ontheir surface IGF-IR but also a great number of Insulin receptor and, asa consequence, a great number of Hybrid-R (Frasca et al., 1999; Sciaccaet al., 1999; Vella et al., 2001).

More particularly, the present invention concerns four differentanti-IGF-IR monoclonal antibodies.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents the in vitro evaluation of anti IGF-1R antibodies inthe MCF-7 model.

FIGS. 2 and 3 represent in vivo evaluation of anti IGF-1R antibodies onDU145.

FIG. 4 represents displacement of [¹²⁵I]-IGF-1 on intact cellsexpressing IGF-IR.

FIG. 5 represents displacement of [¹²⁵I]-IGF-1 on immunocaptured HR-A.

FIG. 6 represents displacement of [¹²⁵I]-IGF-1 on immunocaptured HR-B.

FIG. 7 represents displacement of [¹²⁵I]-INS on intact cells expressingIR-A.

FIG. 8 represents displacement of [¹²⁵I]-INS on intact cells expressingIR-B.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, a subject of the present invention is an isolatedantibody, or one of its functional fragments, said antibody or one ofits said fragments being capable of binding specifically to the humaninsulin-like growth factor I receptor and, if necessary, preferablymoreover capable of inhibiting the natural attachment of the ligandsIGF1 and/or IGF2 of IGF-IR and/or capable of specifically inhibiting thetyrosine kinase activity of said IGF-IR, characterized in that itcomprises a light chain comprising at least one complementaritydetermining region CDR chosen from the CDRs of amino acid sequence SEQID Nos. 1, 2 and 3, or at least one CDR whose sequence has at least 80%,preferably 85%, 90%, 95% and 98% identity, after optimum alignment, withthe sequence SEQ ID Nos. 1, 2 and 3, or in that it comprises a heavychain comprising at least one CDR chosen from the CDRs of amino acidsequence SEQ ID Nos. 4, 5 and 6, or at least one CDR whose sequence hasat least 80%, preferably 85%, 90%, 95% and 98% identity, after optimumalignment, with the sequence SEQ ID No. 4, 5 and 6.

In the present specification and corresponding examples, this antibodywill be referred as 13F5.

In the present description, the terms “to bind” and “to attach” have thesame meaning and are inter-changeable.

In the present description, the terms polypeptides, polypeptidesequences, peptides and proteins attached to antibody compounds or totheir sequence are interchangeable.

It must be understood here that the invention does not relate to theantibodies in natural form, that is to say they are not in their naturalenvironment but that they have been able to be isolated or obtained bypurification from natural sources, or else obtained by geneticrecombination, or by chemical synthesis, and that they can then containunnatural amino acids as will be described further on.

By CDR regions or CDR(s), it is intended to indicate the hypervariableregions of the heavy and light chains of the immunoglobulins as definedby Kabat et al. (Kabat et al., Sequences of proteins of immunologicalinterest, 5th Ed., U.S. Department of Health and Human Services, NIH,1991, and later editions). Three heavy chain CDRs and 3 light chain CDRsexist. The term CDR or CDRs is used here in order to indicate, accordingto the case, one of these regions or several, or even the whole, ofthese regions which contain the majority of the amino acid residuesresponsible for the binding by affinity of the antibody for the antigenor the epitope which it recognizes.

By “percentage of identity” between two nucleic acid or amino acidsequences in the sense of the present invention, it is intended toindicate a percentage of nucleotides or of identical amino acid residuesbetween the two sequences to be compared, obtained after the bestalignment (optimum alignment), this percentage being purely statisticaland the differences between the two sequences being distributed randomlyand over their entire length. The comparisons of sequences between twonucleic acid or amino acid sequences are traditionally carried out bycomparing these sequences after having aligned them in an optimummanner, said comparison being able to be carried out by segment or by“comparison window”. The optimum alignment of the sequences for thecomparison can be carried out, in addition to manually, by means of thelocal homology algorithm of Smith and Waterman (1981) [Ad. App. Math.2:482], by means of the local homology algorithm of Neddleman and Wunsch(1970) [J. Mol. Biol. 48:443], by means of the similarity search methodof Pearson and Lipman (1988) [Proc. Natl. Acad. Sci. USA 85:2444), bymeans of computer software using these algorithms (GAP, BESTFIT, FASTAand TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis., or else by BLAST N or BLAST Pcomparison software).

The percentage of identity between two nucleic acid or amino acidsequences is determined by comparing these two sequences aligned in anoptimum manner and in which the nucleic acid or amino acid sequence tobe compared can comprise additions or deletions with respect to thereference sequence for an optimum alignment between these two sequences.The percentage of identity is calculated by determining the number ofidentical positions for which the nucleotide or the amino acid residueis identical between the two sequences, by dividing this number ofidentical positions by the total number of positions in the comparisonwindow and by multiplying the result obtained by 100 in order to obtainthe percentage of identity between these two sequences.

For example, it is possible to use the BLAST program, “BLAST 2sequences” (Tatusova et al., “Blast 2 sequences—a new tool for comparingprotein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250)available on the site www.ncbi.nlm.nih.gov/gorf/b12.html, the parametersused being those given by default (in particular for the parameters“open gap penalty”: 5, and “extension gap penalty”: 2; the matrix chosenbeing, for example, the matrix “BLOSUM 62” proposed by the program), thepercentage of identity between the two sequences to be compared beingcalculated directly by the program.

By amino acid sequence having at least 80%, preferably 85%, 90%, 95% and98% identity with a reference amino acid sequence, those having, withrespect to the reference sequence, certain modifications, in particulara deletion, addition or substitution of at least one amino acid, atruncation or an elongation are preferred. In the case of a substitutionof one or more consecutive or nonconsecutive amino acid(s), thesubstitutions are preferred in which the substituted amino acids arereplaced by “equivalent” amino acids. The expression “equivalent aminoacids” is aimed here at indicating any amino acid capable of beingsubstituted with one of the amino acids of the base structure without,however, essentially modifying the biological activities of thecorresponding antibodies and such as will be defined later, especiallyin the examples. These equivalent amino acids can be determined eitherby relying on their structural homology with the amino acids which theyreplace, or on results of comparative trials of biological activitybetween the different antibodies capable of being carried out.

By way of example, mention is made of the possibilities of substitutioncapable of being carried out without resulting in a profoundmodification of the biological activity of the corresponding modifiedantibody. It is thus possible to replace leucine by valine orisoleucine, aspartic acid by glutamic acid, glutamine by asparagine,arginine by lysine, etc., the reverse substitutions being naturallyenvisageable under the same conditions.

In a second aspect, a subject of the present invention is an isolatedantibody, or one of its functional fragments, said antibody or one ofits said fragments being capable of binding specifically to the humaninsulin-like growth factor I receptor and, if necessary, preferablymoreover capable of inhibiting the natural attachment of the ligandsIGF1 and/or IGF2 of IGF-IR and/or capable of specifically inhibiting thetyrosine kinase activity of said IGF-IR, characterized in that itcomprises a light chain comprising at least one complementaritydetermining region CDR chosen from the CDRs of amino acid sequence SEQID Nos. 7, 8 and 9, or at least one CDR whose sequence has at least 80%,preferably 85%, 90%, 95% and 98% identity, after optimum alignment, withthe sequence SEQ ID Nos. 7, 8 and 9, or in that it comprises a heavychain comprising at least one CDR chosen from the CDRs of amino acidsequence SEQ ID Nos. 10, 11 and 12, or at least one CDR whose sequencehas at least 80%, preferably 85%, 90%, 95% and 98% identity, afteroptimum alignment, with the sequence SEQ ID Nos. 10, 11 and 12.

In the following specification, this antibody will be referred as 12D5.In a third aspect, a subject of the present invention is an isolatedantibody, or one of its functional fragments, said antibody or one ofits said fragments being capable of binding specifically to the humaninsulin-like growth factor I receptor and, if necessary, preferablymoreover capable of inhibiting the natural attachment of the ligandsIGF1 and/or IGF2 of IGF-IR and/or capable of specifically inhibiting thetyrosine kinase activity of said IGF-IR, characterized in that itcomprises a light chain comprising at least one complementaritydetermining region CDR chosen from the CDRs of amino acid sequence SEQID Nos. 13, 14 and 15, or at least one CDR whose sequence has at least80%, preferably 85%, 90%, 95% and 98% identity, after optimum alignment,with the sequence SEQ ID Nos. 13, 14 and 15, or in that it comprises aheavy chain comprising at least one CDR chosen from the CDRs of aminoacid sequence SEQ ID Nos. 16, 17 and 18, or at least one CDR whosesequence has at least 80%, preferably 85%, 90%, 95% and 98% identity,after optimum alignment, with the sequence SEQ ID No. 16, 17 and 18.

In the following specification, this antibody will be referred as 2D10.

Last, in yet another aspect, a subject of the present invention is anisolated antibody, or one of its functional fragments, capable ofbinding specifically to the human insulin-like growth factor I receptorand, if necessary, preferably moreover capable of inhibiting the naturalattachment of the ligands IGF1 and/or IGF2 of IGF-IR and/or capable ofspecifically inhibiting the tyrosine kinase activity of said IGF-IR,characterized in that it consists in the antibody called 21E3 andregistered at the CNCM as thereafter mentioned.

The antibodies according to the present invention, i.e. 13F5, 12D5, 2D10and 21E3 are preferably specific monoclonal antibodies, especially ofmurine, chimeric or humanized origin, which can be obtained according tothe standard methods well known to the person skilled in the art.

In general, for the preparation of monoclonal antibodies or theirfunctional fragments, especially of murine origin, it is possible torefer to techniques which are described in particular in the manual“Antibodies” (Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor N.Y., pp. 726, 1988) or tothe technique of preparation from hybridomas described by Kohler andMilstein (Nature, 256:495-497, 1975).

The monoclonal antibodies according to the invention can be obtained,for example, from an animal cell immunized against the IGF-IR, or one ofits fragments containing the epitope specifically recognized by saidmonoclonal antibodies according to the invention. Said IGF-IR, or one ofits said fragments, can especially be produced according to the usualworking methods, by genetic recombination starting with a nucleic acidsequence contained in the cDNA sequence coding for the IGF-IR or bypeptide synthesis starting from a sequence of amino acids comprised inthe peptide sequence of the IGF-IR.

The monoclonal antibodies according to the invention can, for example,be purified on an affinity column on which the IGF-IR or one of itsfragments containing the epitope specifically recognized by saidmonoclonal antibodies according to the invention has previously beenimmobilized. More particularly, said monoclonal antibodies can bepurified by chromatography on protein A and/or G, followed or notfollowed by ion-exchange chromatography aimed at eliminating theresidual protein contaminants as well as the DNA and the LPS, in itselffollowed or not followed by exclusion chromatography on Sepharose gel inorder to eliminate the potential aggregates due to the presence ofdimers or of other multimers. In an even more preferred manner, thewhole of these techniques can be used simultaneously or successively.

Chimeric or humanized antibodies are likewise included in antibodiesaccording to the present invention.

By chimeric antibody, it is intended to indicate an antibody whichcontains a natural variable (light chain and heavy chain) region derivedfrom an antibody of a given species in combination with the light chainand heavy chain constant regions of an antibody of a speciesheterologous to said given species.

The antibodies or their fragments of chimeric type according to theinvention can be prepared by using the techniques of geneticrecombination. For example, the chimeric antibody can be produced bycloning a recombinant DNA containing a promoter and a sequence codingfor the variable region of a non-human, especially murine, monoclonalantibody according to the invention and a sequence coding for theconstant region of human antibody. A chimeric antibody of the inventionencoded by such a recombinant gene will be, for example, a mouse-manchimera, the specificity of this antibody being determined by thevariable region derived from the murine DNA and its isotype determinedby the constant region derived from the human DNA. For the methods ofpreparation of chimeric antibodies, it is possible, for example, torefer to the document Verhoeyn et al. (BioEssays, 8:74, 1988).

By humanized antibody, it is intended to indicate an antibody whichcontains CDR regions derived from an antibody of nonhuman origin, theother parts of the antibody molecule being derived from one (or fromseveral) human antibodies. Moreover, some of the residues of thesegments of the skeleton (called FR) can be modified in order toconserve the affinity of the binding (Jones et al., Nature, 321:522-525,1986; Verhoeyen et al., Science, 239:1534-1536, 1988; Riechmann et al.,Nature, 332:323-327, 1988).

The humanized antibodies according to the invention or their fragmentscan be prepared by techniques known to the person skilled in the art(such as, for example, those described in the documents Singer et al.,J. Immun. 150:2844-2857, 1992; Mountain et al., Biotechnol. Genet. Eng.Rev., 10:1-142, 1992; or Bebbington et al., Bio/Technology, 10:169-175,1992). Such humanized antibodies according to the invention arepreferred for their use in in vitro diagnostic methods, or in vivoprophylactic and/or therapeutic treatment.

By functional fragment of an antibody according to the invention, it isintended to indicate in particular an antibody fragment, such as Fv,scFv (sc for single chain), Fab, F(ab′)₂, Fab′, scFv-Fc fragments ordiabodies, or any fragment of which the half-life time would have beenincreased by chemical modification, such as the addition ofpoly(alkylene) glycol such as poly(ethylene) glycol (“PEGylation”)(pegylated fragments called Fv-PEG, scFv-PEG, Fab-PEG, F(ab′)₂-PEG orFab′-PEG) (“PEG” for Poly(Ethylene) Glycol), or by incorporation in aliposome, said fragments having at least one of the characteristic CDRsof sequence SEQ ID Nos. 1 to 6, 7 to 12 or 13 to 18 according to theinvention, and, especially, in that it is capable of exerting in ageneral manner an even partial activity of the antibody from which it isdescended, such as in particular the capacity to recognize and to bindto the IGF-IR, and, if necessary, to inhibit the activity of the IGF-IR.

Preferably, said functional fragments will be constituted or willcomprise a partial sequence of the heavy or light variable chain of theantibody from which they are derived, said partial sequence beingsufficient to retain the same specificity of binding as the antibodyfrom which it is descended and a sufficient affinity, preferably atleast equal to 1/100, in a more preferred manner to at least 1/10, ofthat of the antibody from which it is descended, with respect to theIGF-IR. Such a functional fragment will contain at the minimum 5 aminoacids, preferably 10, 15, 25, 50 and 100 consecutive amino acids of thesequence of the antibody from which it is descended.

Preferably, these functional fragments will be fragments of Fv, scFv,Fab, F(ab′)₂, F(ab′), scFv-Fc type or diabodies, which generally havethe same specificity of binding as the antibody from which they aredescended. According to the present invention, antibody fragments of theinvention can be obtained starting from antibodies such as describedabove by methods such as digestion by enzymes, such as pepsin or papainand/or by cleavage of the disulfide bridges by chemical reduction. Inanother manner, the antibody fragments comprised in the presentinvention can be obtained by techniques of genetic recombinationlikewise well known to the person skilled in the art or else by peptidesynthesis by means of, for example, automatic peptide synthesizers suchas those supplied by the company Applied Biosystems, etc.

More particularly, the invention comprises the antibodies, or theirfunctional fragments, according to the present invention, especiallychimeric or humanized antibodies, obtained by genetic recombination orby chemical synthesis.

According a first approach, the antibody will be define by its heavychain sequence.

In a first preferred manner, the present invention relates to anantibody or one of its functional fragments, according to the invention,characterized in that it comprises a heavy chain comprising at least twoof the three CDRs or the three CDRs of sequence SEQ ID Nos. 4 to 6, orat least two of three CDRs or three CDRs of sequence respectively havingat least 80% identity after optimum alignment with the sequence SEQ IDNos. 4 to 6.

In a second preferred manner, the present invention relates to anantibody or one of its functional fragments, according to the invention,characterized in that it comprises a heavy chain comprising at least twoof the three CDRs or the three CDRs of sequence SEQ ID Nos. 10 to 12, orat least two of three CDRs or three CDRs of sequence respectively havingat least 80% identity after optimum alignment with the sequence SEQ IDNos. 10 to 12.

In a third preferred manner, the present invention relates to anantibody or one of its functional fragments, according to the invention,characterized in that it comprises a heavy chain comprising at least twoof the three CDRs or the three CDRs of sequence SEQ ID Nos. 16 to 18, orat least two of three CDRs or three CDRs of sequence respectively havingat least 80% identity after optimum alignment with the sequence SEQ IDNos. 16 to 18.

According a second approach, the antibody will be now define by itslight chain sequence.

In a likewise preferred first embodiment, the antibody or one of itsfunctional fragments, according to the invention, is characterized inthat it comprises a light chain comprising at least one CDR chosen fromthe CDRs of sequence SEQ ID Nos. 1 to 3, or a CDR whose sequence has atleast 80% identity after optimum alignment with the sequence SEQ ID Nos.1 to 3.

In a second embodiment, the antibody or one of its functional fragments,according to the invention, is characterized in that it comprises alight chain comprising at least one CDR chosen from the CDRs of sequenceSEQ ID Nos. 7 to 9, or a CDR whose sequence has at least 80% identityafter optimum alignment with the sequence SEQ ID Nos. 7 to 9.

In a third preferred embodiment, the antibody or one of its functionalfragments, according to the invention, is characterized in that itcomprises a light chain comprising at least one CDR chosen from the CDRsof sequence SEQ ID Nos. 13 to 15, or a CDR whose sequence has at least80% identity after optimum alignment with the sequence SEQ ID Nos. 13 to15.

According a third approach, the antibody will be now define both by itslight chain sequence and its heavy chain sequence.

In a first preferred manner, the antibody or one of its functionalfragments according to the invention is characterized in that itcomprises a heavy chain comprising the three CDRs of sequence SEQ IDNos. 4 to 6, or three CDRs of sequence having at least 80% of identityafter optimum alignment with the sequence SEQ ID Nos. 4 to 6, and inthat it moreover comprises a light chain comprising the three CDRs ofsequence SEQ ID Nos. 1 to 3, or three CDRs of sequence having at least80% of identity after optimum alignment with the sequence SEQ ID Nos. 1to 3.

In a second preferred manner, the antibody or one of its functionalfragments according to the invention is characterized in that itcomprises a heavy chain comprising the three CDRs of sequence SEQ IDNos. 10 to 12, or three CDRs of sequence having at least 80% of identityafter optimum alignment with the sequence SEQ ID No. 10 to 12 and inthat it moreover comprises a light chain comprising the three CDRs ofsequence SEQ ID Nos. 7 to 9, or three CDRs of sequence having at least80% of identity after optimum alignment with the sequence SEQ ID Nos. 7to 9.

In a third preferred manner, the antibody or one of its functionalfragments according to the invention is characterized in that itcomprises a heavy chain comprising the three CDRs of sequence SEQ IDNos. 16 to 18, or three CDRs of sequence having at least 80% of identityafter optimum alignment with the sequence SEQ ID No. 16 to 18 and inthat it moreover comprises a light chain comprising the three CDRs ofsequence SEQ ID Nos. 13 to 15, or three CDRs of sequence having at least80% of identity after optimum alignment with the sequence SEQ ID Nos. 13to 15.

In yet another preferred embodiment, the antibody or one of itsfunctional fragments according to the invention and called 13F5 ischaracterized in that it comprises a heavy chain of sequence comprisingthe amino acid sequence SEQ ID No. 20, and in that it moreover comprisesa light chain of sequence comprising the amino acid sequence SEQ ID No.19.

In yet another preferred embodiment, the antibody or one of itsfunctional fragments according to the invention and called 12D5 ischaracterized in that it comprises a heavy chain of sequence comprisingthe amino acid sequence SEQ ID No. 22 or 23, and in that it moreovercomprises a light chain of sequence comprising the amino acid sequenceSEQ ID No. 21.

In yet another preferred embodiment, the antibody or one of itsfunctional fragments according to the invention and called 2D10 ischaracterized in that it comprises a heavy chain of sequence comprisingthe amino acid sequence SEQ ID No. 25, and in that it moreover comprisesa light chain of sequence comprising the amino acid sequence SEQ ID No.24.

Another possibility, part of the present invention, is an antibodywherein the three CDRs of the heavy chain are randomly chosen in thegroup comprising the CDRs of each 13F5, 12D5 and 2D10 and wherein thethree CDRs of the light chain are also randomly chosen in the groupcomprising the CDRs of each 13F5, 12D5 and 2D10.

According to another aspect, a subject of the present invention is anantibody or one of its functional fragments, according to the invention,characterized in that it does not attach or it does not attach in asignificant manner to the human insulin receptor IR.

In a preferred manner, said functional fragments according to thepresent invention will be chosen from the fragments Fv, scFv, Fab,(Fab′)₂, Fab′, scFv-Fc or diabodies, or any functional fragment whosehalf-life would have been increased by a chemical modification,especially by PEGylation, or by incorporation in a liposome.

According to another aspect, the invention relates to murine hybridomascapable of secreting monoclonal antibodies according to the presentinvention, especially hybridomas of murine origin such as were depositedat the Centre National de Culture De Microorganismes (CNCM, NationalCenter of Microorganism Cultures) (Institut Pasteur, 25 rue du DocteurRoux, F75724 Paris Cedex 15, France).

The monoclonal antibody here called 13F5, or one of its functionalfragments, characterized in that said antibody is secreted by thehybridoma deposited at the CNCM on Mar. 25, 2004 under the number CNCMI-3193 is, of course, part of the present invention. This hybridomaconsists in a murine hybridoma resulting in the cellular fusion ofimmunized mouse splenocytes with a myeloma cell line (Sp20 Ag14).

The monoclonal antibody here called 12D5, or one of its functionalfragments, characterized in that said antibody is secreted by thehybridoma deposited at the CNCM on Apr. 8, 2004 under the number CNCMI-3195 is, of course, part of the present invention. This hybridomaconsists in a murine hybridoma resulting in the cellular fusion ofimmunized mouse splenocytes with a myeloma cell line (Sp20 Ag14).

The monoclonal antibody here called 2D10, or one of its functionalfragments, characterized in that said antibody is secreted by thehybridoma deposited at the CNCM on 13 May 2004 under the number I-3214is, of course, part of the present invention. This hybridoma alsoconsists in a murine hybridoma resulting in the cellular fusion ofimmunized mouse splenocytes with a myeloma cell line (Sp20 Ag14).

The monoclonal antibody here called 21E3, or one of its functionalfragments, characterized in that said antibody is secreted by thehybridoma deposited at the CNCM on 1 Jul. 2004 under the number I-3249is, of course, part of the present invention. This hybridoma alsoconsists in a murine hybridoma resulting in the cellular fusion ofimmunized mouse splenocytes with a myeloma cell line (Sp20 Ag14).

According to a likewise particular aspect, the present invention relatesto a chimeric antibody, or one of its functional fragments, according tothe invention, characterized in that said antibody moreover comprisesthe light chain and heavy chain constant regions derived from anantibody of a species heterologous to the mouse, especially man, and ina preferred manner in that the light chain and heavy chain constantregions derived from a human antibody are respectively the kappa andgamma-1, gamma-2 or gamma-4 region.

According to a novel aspect, the present invention relates to anisolated nucleic acid, characterized in that it is chosen from thefollowing nucleic acids:

a) a nucleic acid, DNA or RNA, coding for an antibody, or one of itsfunctional fragments, according to the invention;

b) a complementary nucleic acid of a nucleic acid such as defined in a).

By nucleic acid, nucleic or nucleic acid sequence, polynucleotide,oligonucleotide, polynucleotide sequence, nucleotide sequence, termswhich will be employed indifferently in the present invention, it isintended to indicate a precise linkage of nucleotides, which aremodified or unmodified, allowing a fragment or a region of a nucleicacid to be defined, containing or not containing unnatural nucleotides,and being able to correspond just as well to a double-stranded DNA, asingle-stranded DNA as to the transcription products of said DNAs.

It must also be understood here that the present invention does notconcern the nucleotide sequences in their natural chromosomalenvironment, that is to say in the natural state. It concerns sequenceswhich have been isolated and/or purified, that is to say that they havebeen selected directly or indirectly, for example by copy, theirenvironment having been at least partially modified. It is thus likewiseintended to indicate here the isolated nucleic acids obtained by geneticrecombination by means, for example, of host cells or obtained bychemical synthesis.

A hybridization under conditions of high stringency signifies that thetemperature conditions and ionic strength conditions are chosen in sucha way that they allow the maintenance of the hybridization between twofragments of complementary DNA. By way of illustration, conditions ofhigh stringency of the hybridization step for the purposes of definingthe polynucleotide fragments described above are advantageously thefollowing.

The DNA-DNA or DNA-RNA hybridization is carried out in two steps: (1)prehybridization at 42° C. for 3 hours in phosphate buffer (20 mM, pH7.5) containing 5×SSC (1×SSC corresponds to a 0.15 M NaCl+0.015 M sodiumcitrate solution), 50% of formamide, 7% of sodium dodecyl sulfate (SDS),10×Denhardt's, 5% of dextran sulfate and 1% of salmon sperm DNA; (2)actual hybridization for 20 hours at a temperature dependent on the sizeof the probe (i.e.: 42° C., for a probe size >100 nucleotides) followedby 2 washes of 20 minutes at 20° C. in 2×SSC+2% of SDS, 1 wash of 20minutes at 20° C. in 0.1×SSC+0.1% of SDS. The last wash is carried outin 0.1×SSC+0.1% of SDS for 30 minutes at 60° C. for a probe size >100nucleotides. The hybridization conditions of high stringency describedabove for a polynucleotide of defined size can be adapted by the personskilled in the art for oligonucleotides of greater or smaller size,according to the teaching of Sambrook et al. (1989, Molecular cloning: alaboratory manual. 2nd Ed. Cold Spring Harbor).

The invention likewise relates to a vector comprising a nucleic acidaccording to the present invention.

The invention aims especially at cloning and/or expression vectors whichcontain a nucleotide sequence according to the invention.

The vectors according to the invention preferably contain elements whichallow the expression and/or the secretion of the nucleotide sequences ina determined host cell. The vector must therefore contain a promoter,signals of initiation and termination of translation, as well asappropriate regions of regulation of transcription. It must be able tobe maintained in a stable manner in the host cell and can optionallyhave particular signals which specify the secretion of the translatedprotein. These different elements are chosen and optimized by the personskilled in the art as a function of the host cell used. To this effect,the nucleotide sequences according to the invention can be inserted intoautonomous replication vectors in the chosen host, or be integrativevectors of the chosen host.

Such vectors are prepared by methods currently used by the personskilled in the art, and the resulting clones can be introduced into anappropriate host by standard methods, such as lipofection,electroporation, thermal shock, or chemical methods.

The vectors according to the invention are, for example, vectors ofplasmidic or viral origin. They are useful for transforming host cellsin order to clone or to express the nucleotide sequences according tothe invention.

The invention likewise comprises the host cells transformed by orcomprising a vector according to the invention.

The host cell can be chosen from prokaryotic or eukaryotic systems, forexample bacterial cells but likewise yeast cells or animal cells, inparticular mammalian cells. It is likewise possible to use insect cellsor plant cells.

The invention likewise concerns animals, except man, which comprise atleast one cell transformed according to the invention.

According to another aspect, a subject of the invention is a process forproduction of an antibody, or one of its functional fragments accordingto the invention, characterized in that it comprises the followingstages:

a) culture in a medium and appropriate culture conditions of a host cellaccording to the invention; and

b) the recovery of said antibodies, or one of their functionalfragments, thus produced starting from the culture medium or saidcultured cells.

The cells transformed according to the invention can be used inprocesses for preparation of recombinant polypeptides according to theinvention. The processes for preparation of a polypeptide according tothe invention in recombinant form, characterized in that they employ avector and/or a cell transformed by a vector according to the invention,are themselves comprised in the present invention. Preferably, a celltransformed by a vector according to the invention is cultured underconditions which allow the expression of said polypeptide and saidrecombinant peptide is recovered.

As has been said, the host cell can be chosen from prokaryotic oreukaryotic systems. In particular, it is possible to identify nucleotidesequences according to the invention, facilitating secretion in such aprokaryotic or eukaryotic system. A vector according to the inventioncarrying such a sequence can therefore advantageously be used for theproduction of recombinant proteins, intended to be secreted. In effect,the purification of these recombinant proteins of interest will befacilitated by the fact that they are present in the supernatant of thecell culture rather than in the interior of the host cells.

It is likewise possible to prepare the polypeptides according to theinvention by chemical synthesis. Such a preparation process is likewisea subject of the invention. The person skilled in the art knows theprocesses of chemical synthesis, for example the techniques employingsolid phases (see especially Steward et al., 1984, Solid phase peptidesynthesis, Pierce Chem. Company, Rockford, 111, 2nd ed.) or techniquesusing partial solid phases, by condensation of fragments or by aclassical synthesis in solution. The polypeptides obtained by chemicalsynthesis and being able to contain corresponding unnatural amino acidsare likewise comprised in the invention.

The antibodies, or one of their functional fragments, capable of beingobtained by a process according to the invention are likewise comprisedin the present invention.

According to a second embodiment, the present invention concerns anantibody according to the invention such as described further above,characterized in that it is, moreover, capable of binding specificallyto the human epidermal growth factor receptor EGFR and/or capable ofspecifically inhibiting the tyrosine kinase activity of said EGFR.

The invention likewise concerns a pharmaceutical composition comprisingby way of active principle a compound consisting of an antibody, or oneof its functional fragments according to the invention, preferably mixedwith an excipient and/or a pharmaceutically acceptable vehicle.

Another complementary embodiment of the invention consists in acomposition such as described above which comprises, moreover, as acombination product for simultaneous, separate or sequential use, acytotoxic/cytostatic agent and/or an inhibitor of the tyrosine kinaseactivity respectively of the receptors for IGF-I and/or for EGF.

“Simultaneous use” is understood as meaning the administration of thetwo compounds of the composition according to the invention in a singleand identical pharmaceutical form.

“Separate use” is understood as meaning the administration, at the sametime, of the two compounds of the composition according to the inventionin distinct pharmaceutical forms.

“Sequential use” is understood as meaning the successive administrationof the two compounds of the composition according to the invention, eachin a distinct pharmaceutical form.

In a general fashion, the composition according to the inventionconsiderably increases the efficacy of the treatment of cancer. In otherwords, the therapeutic effect of the anti-IGF-IR antibodies according tothe invention is potentiated in an unexpected manner by theadministration of a cytotoxic agent. Another major subsequent advantageproduced by a composition according to the invention concerns thepossibility of using lower efficacious doses of active principle, whichallows the risks of appearance of secondary effects to be avoided or tobe reduced, in particular the effects of the cytotoxic agent.

In addition, this composition according to the invention would allow theexpected therapeutic effect to be attained more rapidly.

In a particularly preferred embodiment, said composition as acombination product according to the invention is characterized in thatsaid cytotoxic/cytostatic agent is chosen from the agents interactingwith DNA, the antimetabolites, the topoisomerase I or II inhibitors, orelse the spindle inhibitor or stabilizer agents or else any agentcapable of being used in chemotherapy. Such cytotoxic/cytostatic agents,for each of the aforesaid classes of cytotoxic agents, are, for example,cited in the 2001 edition of VIDAL, on the page devoted to the compoundsattached to the cancerology and hematology column “Cytotoxics”, thesecytotoxic compounds cited with reference to this document are cited hereas preferred cytotoxic agents.

In a particularly preferred embodiment, said composition as acombination product according to the invention is characterized in thatsaid cytotoxic agent is coupled chemically to said antibody forsimultaneous use.

In a particularly preferred embodiment, said composition according tothe invention is characterized in that said cytotoxic/cytostatic agentis chosen from the spindle inhibitor or stabilizer agents, preferablyvinorelbine and/or vinflunine and/or vincristine.

In order to facilitate the coupling between said cytotoxic agent andsaid antibody according to the invention, it is especially possible tointroduce spacer molecules between the two compounds to be coupled, suchas poly(alkylene) glycols like polyethylene glycol, or else amino acids,or, in another embodiment, to use active derivatives of said cytotoxicagents into which would have been introduced functions capable ofreacting with said antibody according to the invention. These couplingtechniques are well known to the person skilled in the art and will notbe expanded upon in the present description.

In another preferred embodiment, said inhibitor of the tyrosine kinaseactivity of the receptors for IGF-I is selected from the groupconsisting of derived natural agents, dianilinophthalimides, pyrazolo-or pyrrolopyridopyrimidines or else quinazilines. Such inhibitory agentsare well known to the person skilled in the art and described in theliterature (Ciardiello F., Drugs 2000, Suppl. 1, 25-32).

According to yet another embodiment of the invention, the compositionsuch as described above can likewise comprise another antibody compounddirected against the extracellular domain of the HER2/neu receptor, as acombination product for simultaneous, separate or sequential use,intended for the prevention and for the treatment of cancer, especiallythe cancers overexpressing said HER2/neu receptor and the receptorIGF-IR, such as especially cancer of the breast.

Reference can be made especially to the publications of Albanell et al.(J. of the National Cancer Institute, 93(24):1830-1831, 2001) and of Luet al. (J. of the National Cancer Institute, 93(24):1852-1857, 2001)justifying the unexpected interest in combining an anti-HER2/neuantibody with an anti-IGF-IR antibody according to the presentinvention.

In a particular manner, said anti-HER2/neu antibody of the compositionaccording to the invention is the antibody called Trastuzumab (alsocalled Herceptin).

The invention relates, in another aspect, to a composition characterizedin that one, at least, of said antibodies, or one of their functionalfragments, is conjugated with a cell toxin and/or a radioelement.

Preferably, said toxin or said radioelement is capable of inhibiting atleast one cell activity of cells expressing the IGF-IR, in a morepreferred manner capable of preventing the growth or the proliferationof said cell, especially of totally inactivating said cell.

Preferably also, said toxin is an enterobacterial toxin, especiallyPseudomonas exotoxin A.

The radioelements (or radioisotopes) preferably conjugated to theantibodies employed for the therapy are radioisotopes which emit gammarays and preferably iodine¹³¹, yttrium⁹⁰, gold¹⁹⁹, palladium¹⁰⁰,copper⁶⁷, bismuth²¹⁷ and antimony²¹¹. The radioisotopes which emit betaand alpha rays can likewise be used for the therapy.

By toxin or radioelement conjugated to at least one antibody, or one ofits functional fragments, according to the invention, it is intended toindicate any means allowing said toxin or said radioelement to bind tosaid at least one antibody, especially by covalent coupling between thetwo compounds, with or without introduction of a linking molecule.

Among the agents allowing binding in a chemical (covalent),electrostatic or noncovalent manner of all or part of the components ofthe conjugate, mention may particularly be made of benzoquinone,carbodiimide and more particularly EDC(1-ethyl-3-[3-dimethylaminopropyl]-carbodiimide hydrochloride),dimaleimide, dithiobis-nitrobenzoic acid (DTNB), N-succinimidyl S-acetylthio-acetate (SATA), the bridging agents having one or more phenylazidegroups reacting with the ultraviolets (U.V.) and preferablyN-[-4-(azidosalicylamino)butyl]-3′-(2′-pyridyldithio)-propionamide(APDP), N-succinimid-yl 3-(2-pyridyldithio)propionate (SPDP),6-hydrazino-nicotinamide (HYNIC).

Another form of coupling, especially for the radioelements, can consistin the use of a bifunctional ion chelator.

Among these chelates, it is possible to mention the chelates derivedfrom EDTA (ethylenediaminetetraacetic acid) or from DTPA(diethylenetriaminepentaacetic acid) which have been developed forbinding metals, especially radioactive metals, and immunoglobulins.Thus, DTPA and its derivatives can be substituted by different groups onthe carbon chain in order to increase the stability and the rigidity ofthe ligand-metal complex (Krejcarek et al., 1977; Brechbiel et al.,1991; Gansow, 1991; U.S. Pat. No. 4,831,175).

For example diethylenetriaminepentaacetic acid (DTPA) and itsderivatives, which have been widely used in medicine and in biology fora long time either in their free form, or in the form of a complex witha metallic ion, have the remarkable characteristic of forming stablechelates with metallic ions and of being coupled with proteins oftherapeutic or diagnostic interest such as antibodies for thedevelopment of radioimmunoconjugates in cancer therapy (Meases et al.,1984; Gansow et al., 1990).

Likewise preferably, said at least one antibody forming said conjugateaccording to the invention is chosen from its functional fragments,especially the fragments amputated of their Fc component such as thescFv fragments.

The present invention moreover comprises the use of the compositionaccording to the invention for the preparation of a medicament.

More particularly, according to another embodiment, the inventionconcerns the use of an antibody, or one of its functional fragments,and/or of a composition for the preparation of a medicament intended forthe prevention or for the treatment of an illness induced by anoverexpression and/or an abnormal activation of the IGF-I receptor,and/or connected with a hyperactivation of the transduction pathway ofthe signal mediated by the interaction of the 1-IGF1 or IGF2 withIGF-IR.

Preferably, said use according to the invention is characterized in thatthe administration of said medicament does not induce or induces onlyslightly secondary effects connected with inhibition of the insulinreceptor IR, that is to say inhibition of the interaction of the IR withits natural ligands due to the presence of said medicament, especiallyby a competitive inhibition connected with the attachment of saidmedicament to the IR.

The present invention moreover comprises the use of an antibody, or oneof its functional fragments, preferably humanized, and/or of acomposition according to the invention for the preparation of amedicament intended to inhibit the transformation of normal cells intocells with tumoral character, preferably IGF-dependent, especially IGF1-and/or IGF2-dependent.

The present invention likewise relates to the use of an antibody, or oneof its functional fragments, preferably humanized, and/or of acomposition according to the invention for the preparation of amedicament intended to inhibit the growth and/or the proliferation oftumor cells, preferably IGF-dependent, especially IGF1- and/orIGF2-dependent.

In a general manner, a subject of the present invention is the use of anantibody, or one of its functional fragments, preferably humanized,and/or of a composition according to the invention, for the preparationof a medicament intended for the prevention or for the treatment ofcancer preferably expressing IGF-IR and/or of cancer preferably having ahyperactivation of the transduction pathway of the signal mediated bythe interaction of IGF1 or IGF2 with IGF-IR, such as, for example, theoverexpression of IRS1.

The subject of the present invention is likewise the use of an antibody,or one of its functional fragments, preferably humanized, and/or of acomposition according to the invention, for the preparation of amedicament intended for the prevention or for the treatment ofpsoriasis, psoriasis whose epidermal hyperproliferation can be connectedwith the expression or the overexpression of IGF-IR, and/or with thehyperactivation of the transduction pathway of the signal mediated bythe interaction of IGF-IR with its natural ligands (Wraight C. J. etal., Nat. Biotechnol., 2000, 18(5):521-526. Reversal of epidermalhyperproliferation in psoriasis by insulin-like growth factor I receptorantisense oligonucleotides). In another embodiment, an object of theinvention is the use of an antibody, or one of its functional fragments,preferably humanized, and/or of a composition according to theinvention, for the preparation of a medicament intended for theprevention or for the treatment of atherosclerosis.

Among the cancers which can be prevented and/or treated, prostatecancer, osteosarcomas, lung cancer, breast cancer, endometrial cancer orcolon cancer or any other cancer overexpressing IGF-IR is preferred.

According to yet another aspect, a subject of the present invention is amethod of diagnosis, preferably in vitro, of illnesses connected with anoverexpression or an underexpression, preferably an overexpression, ofthe IGF-I receptor starting from a biological sample in which theabnormal presence of IGF-I receptor is suspected, characterized in thatsaid biological sample is contacted with an antibody, or one of itsfunctional fragments, according to the invention, it being possible forsaid antibody to be, if necessary, labeled.

Preferably, said illnesses connected with the overexpression of theIGF-I receptor in said diagnosis method will be cancers.

In another particular embodiment, antibodies according to the inventioncan also be used for the treatment, prevention and/or diagnostic ofillness connected with non only the overexpression of the IGF-IR butalso the overexpression of Hybrid-R.

More particularly, antibody according to the invention is characterizedin that it is also capable of binding to the hybrid-R, isoform(s) Aand/or B, and inhibiting the binding of its native ligands, preferablydesignated herein as IGF1 and/or IGF2 and/or insulin, and/or capable ofspecifically inhibiting the tyrosine kinase activity of said hybrid-R.

Said antibody, or one of its functional fragments, can be present in theform of an immunoconjugate or of a labeled antibody so as to obtain adetectable and/or quantifiable signal.

The antibodies labeled according to the invention or their functionalfragments include, for example, antibodies called immunoconjugates whichcan be conjugated, for example, with enzymes such as peroxidase,alkaline phosphatase, α-D-galactosidase, glucose oxydase, glucoseamylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malatedehydrogenase or glucose 6-phosphate dehydrogenase or by a molecule suchas biotin, digoxygenin or 5-bromodeoxyuridine. Fluorescent labels can belikewise conjugated to the antibodies or to their functional fragmentsaccording to the invention and especially include fluorescein and itsderivatives, fluorochrome, rhodamine and its derivatives, GFP (GFP for“Green Fluorescent Protein”), dansyl, umbelliferone etc. In suchconjugates, the antibodies of the invention or their functionalfragments can be prepared by methods known to the person skilled in theart. They can be coupled to the enzymes or to the fluorescent labelsdirectly or by the intermediary of a spacer group or of a linking groupsuch as a polyaldehyde, like glutaraldehyde, ethylenediaminetetraaceticacid (EDTA), diethylene-triaminepentaacetic acid (DPTA), or in thepresence of coupling agents such as those mentioned above for thetherapeutic conjugates. The conjugates containing labels of fluoresceintype can be prepared by reaction with an isothiocyanate.

Other conjugates can likewise include chemoluminescent labels such asluminol and the dioxetanes, bio-luminescent labels such as luciferaseand luciferin, or else radioactive labels such as iodine¹²³, iodine¹²⁵,iodine¹²⁶, iodine¹³³, bromine⁷⁷, technetium^(99m), indium¹¹¹,indium^(113m), gallium⁶⁷, gallium⁶⁸, ruthenium⁹⁵, ruthenium⁹⁷,ruthenium¹⁰³, ruthenium¹⁰⁵, mercury¹⁰⁷, mercury²⁰³, rhenium^(99m),rhenium¹⁰¹, rhenium¹⁰⁵, scandium⁴⁷, tellurium^(121m), tellurium^(122m),tellurium^(125m), thulium¹⁶⁵, thulium¹⁶⁷, thulium¹⁶⁸, fluorine¹⁸,yttrium¹⁹⁹, iodine¹³¹. The methods known to the person skilled in theart existing for coupling the therapeutic radioisotopes to theantibodies either directly or via a chelating agent such as EDTA, DTPAmentioned above can be used for the radioelements which can be used indiagnosis. It is likewise possible to mention labeling with Na[I¹²⁵] bythe chloramine T method [Hunter W. M. and Greenwood F. C., 1962, Nature194:495] or else with technetium^(99m) by the technique of Crockford etal. (U.S. Pat. No. 4,424,200) or attached via DTPA as described byHnatowich (U.S. Pat. No. 4,479,930).

Thus, the antibodies, or their functional fragments, according to theinvention can be employed in a process for the detection and/or thequantification of an overexpression or of an underexpression, preferablyan overexpression, of the IGF-I receptor in a biological sample,characterized in that it comprises the following steps:

a) the contacting of the biological sample with an antibody, or one ofits functional fragments, according to the invention; and

b) the demonstration of the IGF-IR/antibody complex possibly formed.

In a particular embodiment, the antibodies, or their functionalfragments, according to the invention, can be employed in a process forthe detection and/or the quantification of the IGF-I receptor in abiological sample, for the monitoring of the efficacy of a prophylacticand/or therapeutic treatment of IGF-dependent cancer or else ofpsoriasis or atherosclerosis.

More generally, the antibodies, or their functional fragments, accordingto the invention can be advantageously employed in any situation wherethe expression of the IGF-I receptor must be observed in a qualitativeand/or quantitative manner.

Preferably, the biological sample is formed by a biological fluid, suchas serum, whole blood, cells, a tissue sample or biopsies of humanorigin.

Any procedure or conventional test can be employed in order to carry outsuch a detection and/or dosage. Said test can be a competition orsandwich test, or any test known to the person skilled in the artdependent on the formation of an immune complex of antibody-antigentype. Following the applications according to the invention, theantibody or one of its functional fragments can be immobilized orlabeled. This immobilization can be carried out on numerous supportsknown to the person skilled in the art. These supports can especiallyinclude glass, polystyrene, polypropylene, polyethylene, dextran, nylon,or natural or modified cells. These supports can be either soluble orinsoluble.

By way of example, a preferred method brings into play immunoenzymaticprocesses according to the ELISA technique, by immunofluorescence, orradioimmunoassay (RIA) technique or equivalent.

Thus, the present invention likewise comprises the kits or setsnecessary for carrying out a method of diagnosis of illnesses induced byan overexpression or an underexpression of the IGF-I receptor or forcarrying out a process for the detection and/or the quantification of anoverexpression or of an underexpression of the IGF-I receptor in abiological sample, preferably an overexpression of said receptor,characterized in that said kit or set comprises the following elements:

a) an antibody, or one of its functional fragments, according to theinvention;

b) optionally, the reagents for the formation of the medium favorable tothe immunological reaction;

c) optionally, the reagents allowing the demonstration ofIGF-IR/antibody complexes produced by the immunological reaction.

The invention moreover relates to the use of a composition as acombination product according to the invention, for the preparation of amedicament intended for the prevention or for the treatment of cancer,especially cancers for which said cytotoxic agent or said anti-HER2/neuantibody is generally prescribed and, especially, for which cancers thetumor cells express or overexpress the IGF-I receptor.

A subject of the invention is likewise the use of an antibody accordingto the invention for the preparation of a medicament intended for thespecific targeting of a biologically active compound to cells expressingor overexpressing the IGF-I receptor.

It is intended here by biologically active compound to indicate anycompound capable of modulating, especially of inhibiting, cell activity,in particular their growth, their proliferation, transcription or genetranslation.

A subject of the invention is also an in vivo diagnostic reagentcomprising an antibody according to the invention, or one of itsfunctional fragments, preferably labeled, especially radiolabeled, andits use in medical imaging, in particular for the detection of cancerconnected with the expression or the overexpression by a cell of theIGF-I receptor.

The invention likewise relates to a composition as a combination productor to an anti-IGF-IR/toxin conjugate or radioelement, according to theinvention, as a medicament.

Preferably, said composition as a combination product or said conjugateaccording to the invention will be mixed with an excipient and/or apharmaceutically acceptable vehicle.

In the present description, pharmaceutically acceptable vehicle isintended to indicate a compound or a combination of compounds enteringinto a pharmaceutical composition not provoking secondary reactions andwhich allows, for example, facilitation of the administration of theactive compound(s), an increase in its lifespan and/or in its efficacyin the body, an increase in its solubility in solution or else animprovement in its conservation. These pharmaceutically acceptablevehicles are well known and will be adapted by the person skilled in theart as a function of the nature and of the mode of administration of theactive compound(s) chosen.

Preferably, these compounds will be administered by the systemic route,in particular by the intravenous route, by the intramuscular,intradermal, intraperitoneal or subcutaneous route, or by the oralroute. In a more preferred manner, the composition comprising theantibodies according to the invention will be administered severaltimes, in a sequential manner.

Their modes of administration, dosages and optimum pharmaceutical formscan be determined according to the criteria generally taken into accountin the establishment of a treatment adapted to a patient such as, forexample, the age or the body weight of the patient, the seriousness ofhis/her general condition, the tolerance to the treatment and thesecondary effects noted.

Other characteristics and advantages of the invention appear in thecontinuation of the description with the examples and the figures.

Example 1 Generation of Monoclonal Antibodies Against IGF-1R

Hybridomas were generated by fusion of splenocytes from BALB/c miceimmunized with a soluble α2-β2 heterotetrameric recombinant human IGF-1R(R&D System, Minneapolis, USA) and the SP2/0-Ag14 myeloma cell line. Theresulting murine antibodies were first screened by ELISA and FACSanalysis on MCF-7 cells. Then, a final screen on Sf9-IGF-1r versusSf9-IR cells was performed to eliminate antibodies recognizing bothIGF-1 and IR. The selected MAbs (positives in ELISA and recognizing thewild type receptor on MCF-7 cells) were produced as ascitic fluids andpurified by protein A chromatography before testing either in vitroand/or in vivo as summarized in table 1.

TABLE 1 Selection of anti-IGF-1R monoclonal antibodies

Example 2 In Vitro Activity of Anti-IGF-1R Antibodies

Method

MCF-7 cells from ATCC were routinely cultured in phenol red free-RPMImedium (Invitrogen Corporation, Scotland, UK), 10% FCS (InvitrogenCorporation), 1% L-Glutamine (Invitrogen Corporation). MCF-7 cells wereplated in 96-well tissue culture plates at a density of 5×10⁴ cells/wellin serum-free medium. After 24 hours a dose range of IGF1 from 1 to 50ng/ml was added to the medium either in absence or in presence at afinal concentration of 5 μg/ml of each antibody to be tested. After 3days, cells were pulsed with 0.5 μCi of [³H]thymidine (AmershamBiosciences AB, Uppsala, Sweden) for 16 hours. The magnitude of[³H]thymidine incorporated into trichloroacetic acid-insoluble DNA wasquantified by liquid scintillation counting. Results are expressed as aproliferative index (cpm of cells plus IGF1 plus antibody/cpm of cellsplus antibody alone).

Results

The in vitro evaluation was the first screening of Mabs in terms ofmitogenic activity. For these assays, the generated antibodies, producedas ascitic fluids, were added to MCF-7 cells at the same time as IGF1and compared to the commercially available αIR3 Mab to select antibodiesat least as efficacious as this latter antibody. The positive Mabs (5Mabs) described as (+) in Table 2* of the previous reply are the onegiving proliferative indexes <5 when cells were stimulated with thehighest dose of IGF1 (50 ng/ml). FIG. 1 shows the in vitro activity offour out of the 6 strong in vitro inhibitors (2D10, 12D5, 12B1, 13F5).2F2 and 21E3 Mabs have been considered as a (±)*Mab (5<Proliferativeindex<15 for the highest concentration of IGF1) and 7G3 and 2B10 wereconsidered as non-neutralizing antibodies (proliferative index>15). Itis interesting to notice that the 21E3 is the only Mab of IgG2 isotype.

Example 3 In Vivo Activity of Anti-IGF-1R antibodies

Method

DU145 cells from ATCC were routinely cultured in DMEM medium (InvitrogenCorporation, Scotland, UK), 10% FCS (Invitrogen Corporation), 1%L-Glutamine (Invitrogen Corporation). Cells were split two days beforeengraftment so that they were in exponential phase of growth. Twomillion DU145 cells were engrafted in PBS to Swiss nude mice. One dayafter implantation, animals were divided into groups of 6 mice. Micewere treated s.c. at the opposite of the tumor with 200 μg of eachantibody to be tested, 3-times a week. The control group was eithertreated with a murine isotype control (EC2) in the first screening orPBS for subsequent screenings as it has been shown in the firstexperiment that no difference in tumor growth was observed between these2 groups of mice. Tumor volume was measured once a week and calculatedby the formula: π/6×length×width×height.

Results

Three in vivo experiments were performed to test a panel of Mabs. FIGS.2 and 3 show that 13F5, 2D10 and 6E5 significantly inhibit the in vivogrowth of DU 145 cells. Statistical analysis (Mann and Whitney test) areshown in Table 2.

TABLE 2 Statistical analysis of in vivo data First screening D16 D23 D29D36 D43 PBS/13F5 Mann-Whitney p = 0.47 p = 0.086 p = 0.046 p = 0.015 p =0.023 (Wilcoxon) Second screening D12 D20 D26 D30 PBS/2D10 Mann-Whitneyp = 0.0041 p = 0.0017 p = 0.0027 p = 0.0027 (Wilcoxon) PBS/6E5Mann-Whitney p = 0.027 p = 0.013 p = 0.0092 p = 0.019 (Wilcoxon)PBS/12B1 Mann-Whitney p = 0.11 p = 0.11 p = 0.067 p = 0.11 (Wilcoxon)PBS/2F2 Mann-Whitney p = 0.18 p = 0.067 p = 0.050 p = 0.14 (Wilcoxon)Third screening D12 D20 D26 D33 PBS/16A12 Mann-Whitney p = 0.063 p =0.087 p = 0.19 p = 0.11 (Wilcoxon)

Example 4 Evaluation of 2D10, 12D5, 13F5 Ability to Bind to IGF-IR andHybrid-R

The used cells for this study are listed thereafter:

R+: R− fibroblasts stably transfected with the IGF-I receptor (IGF-IR)cDNA

R−/IR-A: R− fibroblasts stably transfected with the insulin receptorisoform A (IR-A) cDNA

R−/IR-B: R− fibroblasts stably transfected with the insulin receptorisoform B (IR-B) cDNA

R+/IR-A: R− fibroblasts stably co-transfected with the IGF-I and theinsulin receptor isoform A cDNA and, therefore, expressing hybridreceptors A (Hybrid-RsA)

R+/IR-B: R− fibroblasts stably co-transfected with the IGF-I and theinsulin receptor isoform B cDNA and, therefore, expressing hybridreceptors A (Hybrid-RsB).

Example 4-1 Displacement Analysis of [¹²⁵I]IGF1 on IGF-IR by 2D10, 12D5,13F5 and αIR-3

[¹²⁵I]IGF-1 (20,000 cpm) was allowed to bind to R+ intact cells for 16hours at 4° C., in the absence or presence of increasing concentrationsof unlabeled ligand (IGF1, IGF2 or insulin) or antibodies (2D10, 12D5,13F5). Results are plotted as percent of maximal specific binding andare represented on FIG. 4.

Both 2D10 and 13F5 efficaciously and fully displaced IGF1 withsub-nanomolar affinities and in this example with an IC₅₀ of 0.15 and0.20 nM, respectively, as compared to the reference antibody αIR3 (IC₅₀:0.05 nM). The affinities are higher than those of the natural IGF-IRligands IGF1 (2.2 nM in this example) and IGF2 (15 nM in this example).

Example 4-2 Displacement Analysis of [¹²⁵I]IGF1 on Hybrid-RsA by 2D10,12D5, 13F5 and 47-9

Hybrid-RsA from R+/IR-A cell lysates were immunocaptured in Maxisorbplates coated with anti IR antibody 83-7.

[¹²⁵I]-IGF1 (FIG. 5) was then allowed to bind to immunocapturedreceptors in the absence or the presence of increasing concentrations ofunlabeled ligand (IGF1, IGF2 or insulin) or antibodies (2D10, 12D5,13F5, 47-9, 9G4). Results are plotted as percent of maximal specificbinding and are represented in FIG. 5.

2D10 and 13F5 displaced efficaciously and fully labeled IGF1 with verysimilar subnanomolar affinities, and in this example of 0.2 and 0.35 nMrespectively. By comparison, 47-9 yielded an IC₅₀ value of 0.18 nM (FIG.5).

These affinities are higher than those of the natural Hybrid-RsA ligandsIGF1 (2.0 nM in this example) and IGF2 (12 nM in this example).

Example 4-3 Displacement Analysis of [¹²⁵I]IGF1 on Hybrid-RsB by 2D10,12D5, 13F5 and 47-9

Hybrid-RsB from R+/IR-B cell lysates were immunocaptured in Maxisorbplates coated with 83-7 antibody.

[¹²⁵I]-IGF1 (FIG. 6) was then allowed to bind to immunocapturedreceptors in the absence or the presence of increasing concentrations ofIGF1, IGF2, insulin or antibodies (2D10, 12D5, 13F5, 47-9, 9G4). Resultsare plotted as percent of maximal binding.

2D10 and 13F5 displaced efficaciously and fully labeled IGF1 with verysimilar subnanomolar affinities, and in this example of 0.04 and 0.15respectively. By comparison, 47-9 was less effective with an IC₅₀ valueof 0.40 nM (FIG. 6).

Example 4-4 Displacement Analysis of [¹²⁵I]Insulin on Insulin Receptor A(IR-A) and B (IR-B) Isoforms by 2D10, 12D5, 13F5 and MA-10

[¹²⁵I]insulin (40,000 cpm) was allowed to bind to R⁻/IR-A or R⁻/IR-Bintact cells for 16 hours at 4° C., in the absence or presence ofincreasing concentrations of unlabeled ligand (IGF1, IGF2 or insulin) orantibodies (2D10, 12D5, 13F5). Results are plotted as percent of maximalspecific binding and are represented on FIGS. 7 and 8, respectively forIR-A and IR-B.

Neither 2D10, nor 12D5 nor 13F5 displaced insulin, in contrast to thereference antibody MA-10 (IC₅₀: 0.90 and 1.5 nM for IR-A (FIG. 7) andIR-B (FIG. 8), respectively).

1-27. (canceled)
 28. An isolated antibody or binding fragment thereofcapable of binding to the human insulin-like growth factor I receptor(IGF-IR), wherein the antibody or binding fragment thereof comprises alight chain and a heavy chain, wherein the light chain comprises thethree complementarity determining regions (CDRs) of sequence SEQ ID NO:7, 8 and 9; and the heavy chain comprises the three CDRs of sequence SEQID NO: 10, 11 and 12 or the three CDRs of sequence SEQ ID NO: 10, 11 and12 wherein in the sequence SEQ ID NO:11 the arginine residue located inposition 6 is a glycine residue.
 29. The antibody or binding fragmentthereof of claim 28, wherein the antibody is called 12D5 and comprises aheavy chain amino acid sequence of SEQ ID NO: 22 or 23 and a light chainamino acid sequence of SEQ ID NO:
 21. 30. A murine hybridoma capable ofsecreting the antibody as claimed in claim 28 which has been depositedat the CNCM, Institut Pasteur, Paris, on Apr. 8, 2004 under the numberI-3195.
 31. An antibody secreted by the hybridoma as claimed in claim 30or a binding fragment thereof.
 32. The antibody or binding fragmentthereof of claim 28, wherein the antibody is a chimeric antibody.
 33. Acomposition comprising the antibody or binding fragment thereof asclaimed in claim 28 and a pharmaceutically acceptable carrier.
 34. Acomposition comprising the antibody produced by the hybridoma of claim30 or the binding fragment thereof and a pharmaceutically acceptablecarrier.
 35. The composition as claimed in claim 33, further comprisinga cytotoxic agent, a cytostatic agent, a tyrosine kinase inhibitor ofIGF-I receptor, and/or a tyrosine kinase inhibitor of EGF receptor.